Project Summary The myosin super-relaxed (SRX) state, first described six years ago, is emerging as an important player in muscle regulation and function, yet its mechanistic structure and dynamics have not been resolved. The goal of this proposal is to incorporate time-resolved fluorescence resonance energy transfer (TR-FRET) with quantitative epifluorescence microscopy of fluorescent nucleotides, to detect and quantitate the SRX in chemically skinned skeletal muscle fibers from different disease mouse models. The long-term goal is to establish the role of SRX in age-related attenuation of muscle function, and use these insights to design therapeutic approaches. The population of SRX myosin in resting skeletal muscle is as much as 50%. These molecules are sequestered on the myosin thick-filament backbone and are thus auto-inhibited, catalyzing ATP hydrolysis about 10 times slower than purified myosin in solution, while generating no force. My proposal focuses on the role of SRX in the attenuation of muscle function with age, focusing on three independent but inter-related Aims: concerning the roles of sex-specificity (Aim 1), muscle thermogenesis and thus control of whole body basal metabolic rate (Aim 2), and metabolic diseases such as obesity and diabetes (Aim 3). Thus, the myosin SRX provides a platform for potential innovative treatments for obesity and diabetes. However, the molecular mechanism by which this SRX state affects aging muscle physiology and pathophysiology is unresolved. To solve this problem, my effort will be focused on applying site-directed spectroscopic probes on myosin to elucidate its structural changes as it undergoes transitions between the super-relaxed state and the normal relaxed state. The SRX can be rapidly mobilized into the normal relaxed or the active state in response to regulatory allosteric cues such as increased in cellular calcium, regulatory light chain (RLC) phosphorylation, and decreased temperature. These perturbations will be thoroughly tested on each proposed mouse model (Aims 1-3). This will be the first time that the molecular dynamics of SRX myosin will be directly studied through the physiological lens of aging and its associated metabolic disorders. Results from this work will provide crucial insights into the nature of the aging process on skeletal myosin and map a path toward novel therapies to treat diseases and extend healthy, active years of life by targeting myosin. The present fellowship proposal will also provide extensive training opportunity in muscle biochemistry, biophysics, and physiology, contributing to a meaningful MD/PhD degree. The training will prepare me to become an independent biomedical researcher with the means to apply optical spectroscopy to translational science, to both appreciate the molecular basis of disease and subsequent development of new treatments. This work aligns strongly with the mission statement of the NIA, by increasing the molecular understanding of the biological processes on aging along with fostering the development of a physician-scientist.